A cDNA sequence encoding a Drosophila Ace-paralogous acetylcholinesterase (AChE) precursor of 701 amino acid residues was identified as the second AChE gene (Ace2) transcript from Culex tritaeniorhynchus. The Ace2 gene is tightly linked to organophosphorus insecticide (OP)-insensitivity of AChE on chromosome 2. The cDNA sequences were compared between an insecticide-susceptible strain and the resistant strain, TYM, that exhibits a 870-fold decrease in fenitroxon-sensitivity of AChE. Two amino acid substitutions were present in TYM mosquitoes. One is F455W whose homologous position in Torped AChE (Phe331) is located in the vicinity of the catalytic His in the acyl pocket of the active site gorge. The other substitution is located to a C-terminal Ile697 position that apparently seems to be excluded from the mature protein and is irrelevant to catalytic activity. The F455W replacement in the Ace2 gene is solely responsible for the insecticide-insensitivity of AChE in TYM mosquitoes.